Titration method

ABSTRACT

The invention relates to a titration method for extremely small quantities of liquid wherein a drop of an analyte held together by its surface tension is applied to the substantially flat surface of a solid, preferably a solid chip, a quantity of the titrant which is smaller than the quantity of the analyte drop, is brought in contact with the analyte drop for the reaction and a characteristic quantity for the reaction between titrant and analyte is measured during and/or after the reaction.

The invention relates to a titration method for liquids wherein ananalyte is brought in contact with a titration quantity of a titrant anda parameter which changes during the reaction between the titrant andanalyte is studied.

In biology and chemistry, extremely small quantities of liquid mustfrequently be analysed in this way. Possible measured quantities forexample are the pH of the liquid, the concentration of molecules havingoxidising or reducing groups or also of heavy metals.

Frequently, only extremely small quantities of the materials to bestudied are available. In order to be able to make quantitative studies,the titrant must be supplied to the analyte in very small quantities inorder: to be able to investigate as accurately as possible the change inparameters of the mixture caused by adding the titrant as a function ofthe quantity of titrant. If only a small amount of material isavailable, for accurate titration many drops of small volume of thetitrant must be produced reproducibly and successively combined with theanalyte. With increasingly smaller volumes, the flows are increasinglylaminar. Thus, at small volumes mixing of the analyte with the titrantis found to be increasingly difficult.

The object of the present invention is to provide a titration methodthat is reproducible even with extremely small volumes of liquid in therange between one nanolitre and a few microlitres and with whichreliable titration can be performed.

This object is solved using a titration method having the features ofclaim 1. The dependent claims are directed towards advantageousembodiments.

According to the invention a drop of the analyte held together by itssurface tension is applied to a substantially flat surface of a solid. Atitration quantity of the titrant is brought in contact with the analytedrop, wherein the quantity of the titrant is smaller than the quantityof the analyte drop. During or after the reaction a characteristicquantity for the reaction between titrant and analyte is measured. Ifnecessary, another small titration quantity of the titrant is brought incontact with the analyte, in order to determine the change in themeasured quantity with increasing quantity of titrant.

For the purposes of the present document the term “solid” designatesboth solids of crystalline material, e.g. LiNbO₃ or quartz, as well asstructures made of other materials, e.g. plastic.

Both titrant and analyte can comprise among other things pure liquids,mixtures, dispersions or suspensions as well as liquids in which solidparticles are located. Likewise, the titrant or the analyte can containbiological material, e.g., cells, macromolecules, proteins, antibodies,antigens or DNA.

In the method according to the invention, the analyte is a single dropwhich is held together by its surface tension. There is no need for anyreaction vessels which could negatively influence the titration, forexample, by adhesion. There is no edge interaction with any side wallsof the vessel and it is possible to titrate very small quantities ofliquid.

The method according to the invention allows macroscopic titration to beminiaturised by several orders of magnitude. With a limited quantity ofsample, the concentration can be very much higher so that the methodaccording to the invention is suitable for detecting extremely smallquantities of samples or for analysing extremely small volumes. As aresult of the small volumes in the range of a few nanolitres, thediffusion lengths are small and the reaction times are short.

The measured quantity can, for example, be the conductivity, the pH orthe reaction heat which vary when the titrant is added to the analyte.Furthermore, an indicator can be dissolved in the analyte which, forexample, brings about a colour change. At a certain concentration ratiobetween titrant and analyte in the analyte drop, a colour change isbrought about by this indicator. Other measured quantities known frommacroscopic titration can also be used.

The method according to the invention is preferably carried out on asolid chip, as is known, for example, from semiconductor technology.Such chips can be processed very simply using known techniques and allowelectrodes or functionalised layers, for example, to be applied usingknown lithographic techniques. Such chip units can be used as part of“lab-on-the-chip” technology (see O. Müller, Laborwelt 1/2000, pages36-38) in the miniaturisation of chemical and biological processes. Aplurality of analysis stations can be arranged on such a chip with whichthe titration method according to the invention can be carried out orother analytical steps can be performed. In addition, integration withother units of a lab-on-the-chip can easily be achieved.

If the titration quantity of the titrant is applied to the surface, forexample, using a pipetting robot or a piezodispenser, the small quantityof the analyte drop is already partly mixed by the impact of the titresolution on the analyte. In order to promote the reaction betweentitrant and analyte or mix liquid located at an analysis point, asurface acoustic wave is advantageously launched in the direction of theanalysis point during the reaction between analyte and titrant. Themomentum transfer of a surface acoustic wave sets the liquid on thesurface in motion and results in its thorough mixing. In this case, themomentum of the surface acoustic wave is brought about by the mechanicaldeformation of the surface or by the interaction of changes in theelectric field caused by the mechanical deformation of the surface withcharged or polarisable particles which may be present in the liquid.

The titrant can be brought in contact with the analyte drop in dropsusing a pipetting robot or piezodispenser. However, it is especiallysimple and advantageous if drops of the titrant are moved on the surfaceof the solid itself in the direction of the analyte. The movement of thetitration quantity towards the analyte drop can also be triggered withthe aid of a surface acoustic wave. The movement on the surface by themomentum transfer of a surface acoustic wave makes it possible toachieve a particularly directional and defined movement. The suitablefrequency of the surface acoustic wave depends on the diameter of thedrop to be moved and can be determined, for example, in preliminaryexperiments.

The surface acoustic waves advantageously used for mixing the liquid atthe analysis point and/or for moving the titration quantity of thetitrant towards the analysis point can be generated using one or aplurality of interdigital transducers on a piezoelectric solid surface,whose direction of emission preferably corresponds to the direction ofthe desired momentum transfer. Such a piezoelectric surface can, forexample, be formed from an LiNbO₃ or quartz crystal. Equally, apiezoelectric coating, e.g. ZnO, comprising another material can beprovided. The surface can also be provided with a sufficiently thinbiocompatible protective layer. In general, the use of interdigitaltransducers to generate surface acoustic waves to move small quantitiesof liquid is described in DE-A-100 55 318.

In a simple arrangement of this preferred embodiment, a drop of thetitrant is applied to the solid surface, which drop is held together byits surface tension. For the titration the small quantity of the titrantis removed from this drop and supplied to the analyte drop at theanalysis point wherein this small titration quantity of the titrantmoves on the surface. This guarantees a higher reproducibility of thedrop size than when using a known dispenser and a higher accuracy ofimpact of the titration quantity on the analyte drop.

The analyte is advantageously brought onto a specially functionalisedanalysis point on the solid surface whose area is more strongly wettedby the analyte than the surrounding solid surface. Such an analysispoint holds the drop of analyte at a predetermined point so that anyflowing apart or drifting away of the analyte is prevented.

The titrant drop serving as a reservoir from which the small titrationquantity of the titrant supplied to the analyte drop is removed can belocated on an anchor point on the surface of the solid, which is betterwetted with the titrant liquid that its surrounding solid surface. Inthis way, it is ensured that the titrant remains at a certain point onthe surface and does not leave this without the action of an externalforce.

The titration quantity of the titrant which is supplied to the analytecan advantageously be moved on the solid surface along a path whosesurface is better wetted with the titrant than its surrounding surface.The titration quantity preferably moves on this path so that acontrolled movement is ensured. Such a path can be achieved, forexample, by modulation of the wetting properties as is described for themovement of quantities of liquids on surfaces in DE-A-100 55 318.

In order to separate a small quantity of the titrant from the reservoirdrop at the anchor point, the reservoir drop at the anchor point can beguided over a path which is connected to the anchor point and/or theanalysis point wherein the connection comprises a region which is sonarrow that as a result of its surface tension the reservoir drop at theanchor point cannot leave the anchor point without the action of anexternal force. If the reservoir drop is driven on this path to thisnarrow point by the action of an external force, it breaks away in adefined fashion.

Alternatively, a reservoir drop can also be moved on the surface by thetransfer of momentum, for example, over one or a plurality of smallsurface part regions, which are more strongly wetted by the titrantliquid than their surroundings. The area of this surface part region isselected to be so small that it is smaller than the contact area of thedrop with the surface. If the reservoir drop is guided once or manytimes over such surface part regions, a small quantity of the titrantremains at these retaining points and can be moved towards the analytedrop for titration. Thus, a small titration quantity can be separated ina very simple and reproducible fashion.

In order to prevent the small quantities of liquid from vaporising toorapidly, the titration method is advantageously carried out in aclimatic chamber to maintain defined thermodynamic boundary conditions.

The method according to the invention makes it possible to miniaturisethe macroscopic titration. Volumes in the range of a few nanolitres toseveral microlitres can be titrated. In particular, when using surfaceacoustic waves, in addition to movement of the titrant on the surfacethe surface acoustic wave can be used for thorough mixing to make thetitration result more reproducible.

Methods of analysis such as Scintillation Proximity Assay (SPA) orFluorescence Resonance Energy Transfer (FRET) such as are described inJ. Osborn, Life Science News, March 2001, pages 1-4, “A review ofradioactive and non-radioactive-based techniques used in life scienceapplications—Part II High-throughput screening”, can also be carried outparticularly advantageously using the method according to the invention.

The invention is explained in detail with reference to particularembodiments which are shown schematically and not to scale in theappended drawings. In the figures:

FIG. 1 shows the implementation of the titration method according to theinvention,

FIG. 2 shows another embodiment of the titration method according to theinvention,

FIG. 3 shows a further embodiment of the titration method according tothe invention and

FIG. 4 shows a process step in a preferred embodiment of the titrationmethod according to the invention.

FIG. 1 shows a solid chip, e.g. a piezoelectric lithium niobate chip 5,on whose surface 7 the titration method according to the invention canbe carried out. A drop 1 of an analyte in the order of magnitude of. 0.5nl to 100 nl is located on an analysis point 15 whose wetting propertiesdiffer from those of its surroundings. If the analyte comprises, forexample, an aqueous solution, the analysis point 15 is hydrophiliccompared to the surrounding solid surface. This can be achieved forexample by the surrounding surface being made hydrophobic bysilanisation. An analysis point with an area of, for example 100 μm x100 μm is suitable for a typical quantity of liquid of 0.5 nl to 10 nl.

A reservoir drop 3 of the titrant solution is located at an anchor point16. The anchor point 16 is also arranged such that it is more stronglywetted with the titrant solution than the surrounding solid surface.

The analysis point 15 and anchor point 16 are interconnected via a path18 which also exhibits such wetting properties that it is better wettedwith the titrant solution than the surrounding solid surface. The path18 is constricted at the narrow points 14, 12 such that as a result ofits surface tension, the drop located at the anchor point 16 or theanalysis point 15 cannot leave the analysis point 15 or the anchor point16 without the action of an external force.

9, 11 and 13 denote interdigital transducers which are suitable forexciting surface acoustic waves on the surface 7 of the lithium niobatecrystal 5. In their simplest form the interdigital transducers consistof two electrodes with finger-like intermeshing continuations. Applyingan alternating field of the order of magnitude of 100 MHz to theelectrodes of one interdigital transducer results in excitation of asurface acoustic wave having a wavelength corresponding to the fingerspacing of the finger-like intermeshing electrodes and whose directionof propagation is substantially perpendicular to the finger electrodes.In the case of the interdigital transducer 9, this is indicatedschematically for example by the arrow 10. Each transducer comprises alarge number of intermeshing fingers of which respectively only a feware shown schematically and not to scale. Other transducer geometriescan also be used, as are known from the technology of the surfaceacoustic wave filter.

The interdigital transducers 9 are aligned such that a surface acousticwave excited by them moves towards the analysis point 15. Theinterdigital transducer 11 produces a surface acoustic wave in thedirection 19. The interdigital transducer 13 ultimately produces asurface acoustic wave in the direction 21. The electrical connections tothe electrodes of the interdigital transducers which are provided in aconventional fashion are not shown for purposes of clarity.

In a schematic representation 23 shows the tip of an inherently knownpiezodispenser for applying the reservoir drop 3 of the titrant to theanchor point 16. The outflow of liquid from the dispenser tip 23 isindicated by the arrow 24.

Using the apparatus shown the method according to the invention can becarried out as follows.

First, a drop of the analyte 1 is applied to the analysis point 15 usinga dispenser head tip not shown, which is similar to the dispenser tip23. As a result of the specially selected wetting properties of theanalysis point 15 compared with the wetting properties of thesurrounding solid surface 7, the drop 1 which is held together by itssurface tension does not leave the analysis point 15. A drop of thetitrant 3 is applied to the anchor point 16 using the dispenser tip 23.Likewise as a result of its surface tension and the wetting propertiesof the anchor point compared to the wetting properties of thesurrounding solid surface 7 (e.g. hydrophilic compared to thesurrounding solid surface in the case of an aqueous titrant solution),this drop 3 does not leave the anchor point 16. The applied volumes ofthe analyte or the titrant can be in the range of one picolitre toseveral 100 microlitres.

An alternating frequency, e.g. a few 100 MHz is now applied to theinterdigital transducer 13 so that a surface acoustic wave is generatedin the direction 21. The momentum transfer of the surface acoustic wavemoves the drop 3 in the direction of the narrow point 14 which connectsthe anchor point 16 to the path 18. A small quantity of the drop 3 movesover the narrow point 14 and breaks away in a defined fashion withsuitable dimensioning. The required reduction in the width of the narrowpoint 14 can be determined, for example, by preliminary experiments. Theseparated quantity of titrant can be a few nanolitres example but shouldbe less than about one tenth of the quantity of analyte at the analysispoint 15.

The withdrawn part 17 of the titrant, the titration quantity, is alsomoved away from the anchor point 16 by momentum transfer of the surfaceacoustic wave generated using the interdigital transducer 13. Themovement of the small titration quantity of the titrant towards theanalysis point 15 is continued using a second transducer 11.

The small titration quantity 17 meets the analyte drop 1 at the analysispoint 15. The reaction between titrant and analyte can be accelerated byusing a surface acoustic wave generated by one of the interdigitaltransducers 9. The surface acoustic wave can be detected after passingthrough the analysis point 15 using a second interdigital transducer 9.As a result of the reaction of the analyte with the titrant, theattenuation of the surface acoustic wave may have changed for example sothat information on the reaction can be obtained. In addition, bycomparison with corresponding reference measurements the precisequantity of the analyte at the analysis point 15 can be determined fromthe attenuation of the surface acoustic wave.

In another embodiment of the method a surface acoustic wave can belaunched towards the analysis point 15 from each of the interdigitaltransducers 9 in order to accelerate the reaction or effectively mix theliquid.

By means of a suitably pulsed surface acoustic wave generated using theinterdigital transducer 13, a plurality of small titrant quantities 17can be moved in a defined fashion towards the analysis point 15 in themanner described to carry out a titration 1.

A plurality of suitable devices can be provided in parallel on a chip sothat a plurality of experiments can be carried out in parallel.

Among other things, ICT (Isothermal Calorimetric Titration) or DSC(Differential Scanning Calorimetry) can be carried out using the methodaccording to the invention as described in a review article by I.Jelesarov and H. R. Bosshard in J. Mol. Recognit. 1999; 12: 3-18.

An alternative method is shown in FIG. 2. For the sake of clarity FIG. 2does not show the transducer 9 which may be provided for thoroughmixing. The same reference numbers denote otherwise the same elements asin FIG. 1. In the method shown in FIG. 2, the conductivity of theanalyte 1 is measured for the titration. For this purpose, electrodes 25connected to the analysis point 15 are provided on the surface 7 of thesolid chip 5. Electrical connections lead to a conductivity measuringdevice 27. By successively adding titrant in small quantities 17 theconductivity of the analysis drop 1 changes, which can be determinedusing the conductivity measuring device 27. In order to carry out such aprocess sequence according to the invention, the analysis point 15 ismade of a non-conductive material.

FIG. 3 shows another embodiment of the method according to theinvention. An optical measurement is made instead of the conductivitymeasurement from FIG. 2. Again the transducers 9 which may be providedfor thorough mixing are not shown. A light-emitting diode 31 or anothersuitable light source illuminates the solid chip 5 from below. Forexample, the optical signal is intercepted using a glass fibre 29 andpassed on to an evaluation device not shown and evaluated in aninherently known fashion. Using this embodiment, for example, it ispossible to measure the colour change of an analyte in which anindicator is dissolved which changes colour after adding a certainquantity of titrant. If a non-transparent substrate is used, the lightpath can also be selected as parallel to the surface 7 of the chip 5.

The embodiments of the method according to the invention described usethe narrow point 14 in order to withdraw a defined quantity 17 of thetitrant from the titrant 3. FIG. 4 shows an alternative possibility forseparating a small quantity of titrant. The same reference numbersdenote comparable elements as in FIGS. 1 to 3. A reservoir drop 3 islocated on an anchor point 16. As a result of momentum transfer of asurface acoustic wave generated using the interdigital transducer 13closest to the corresponding anchor point 16, the reservoir drop 3 isdriven towards the second anchor point 16. The momentum transfer ofanother surface acoustic wave generated using the interdigitaltransducer 13 which is closest to the second anchor point drives thereservoir drop back again. In this case, said drop moves to and froalong the indicated section 43. It crosses a surface region 41 whosearea is smaller than the contact area of the reservoir drop 3 with thesolid surface 7 once or many times. This surface region 41 has suchwetting properties that the liquid of the reservoir drop 3 wets thismore strongly that its surrounding solid surface. After crossing thesurface region 41 once or several times, a small titrant quantity 17 hasseparated from the reservoir drop 3. In the case of an aqueous titrantsolution, the surface region 41 has hydrophilic properties for example.

After the small titrant quantity 17 has been separated from thereservoir drop 3 in this manner, with the aid of the momentum transferof a surface acoustic wave which can be generated for example indirection 45 using an interdigital transducer 11, the titrant quantity17 can be moved away from the surface region 41, e.g. towards ananalysis point not shown in FIG. 4, in order to be combined there withthe analyte drop, as has been described above with reference to FIGS. 1to 3.

The reservoir drop 3 and the titrant drop 17 can in this case be movedalong preferably wetted paths as have already been described withreferences to FIGS. 1 to 3 and are denoted there by the reference number18. Such paths advantageously have a lateral expansion which is smallerthan the diameter of the surface region 41. The method according to theinvention and the separation of the small titrant quantity 17 describedcan however be carried out without such paths so that these are notshown in FIG. 4.

Using the method shown in FIG. 4, for example 20-picolitre smalldroplets 17 can be separated from a 50-nanolitre reservoir drop 3. Aplurality of surface regions 41 can be provided along the path of thereservoir drop 3 if a plurality of titrant quantities 17 are to beseparated simultaneously. Depending on the property of the liquid to bemanipulated in the reservoir drop 3, suitable geometries for the surfaceregion 41, e.g. circular or annular, can be determined by suitablepreliminary tests.

The method described in FIG. 4 for separating a small titrant quantity17 from a reservoir drop 3 can naturally also be combined with all theembodiments for the titration and the subsequent analysis which havealready been described above.

1. A titration method for small quantities of liquid wherein a) a drop(1) of the analyte held together by its surface tension is applied to asubstantially flat surface (7) of a solid, preferably a solid chip (5),b) a titration quantity (17) of the titrant, which is smaller than thequantity of the analyte drop (1), is brought in contact with the analytedrop (1) for the reaction, c) a characteristic quantity for the reactionbetween titrant and analyte is measured during and/or after thereaction, and d) if necessary, steps b) and c) are repeated.
 2. Thetitration method according to claim 1, wherein during the reactionbetween analyte and titrant a surface acoustic wave is launched in thedirection (10) of the analyte drop to promote the reaction betweentitrant and analyte or to thoroughly mix the analytes and titrants. 3.The titration method according to claim 2, wherein the titrationquantity (17) of the titrant on the solid surface (7) is moved towardsthe analyte (1).
 4. The titration method according to claim 3, wherein areservoir drop (3) of the titrant is brought onto the solid surface (7)which is held together as a result of its surface tension and thetitration quantity (17) of the titrant is separated from this reservoirdrop (3) for titration and is supplied to the analyte drop (1).
 5. Thetitration method according to claim 4, wherein the movement of thetitration quantity (17) is triggered by the momentum transfer of asurface acoustic wave.
 6. The titration method according to claim 2,wherein one or a plurality of interdigital transducers (9, 11, 13) on apiezoelectric solid surface (7) are used to generate the surfaceacoustic wave or surface acoustic waves with a direction of emission ofthe surface acoustic waves in the direction of the desired momentumtransfer.
 7. The titration method according to claim 4, wherein the drop(1) of the analyte is brought onto an analysis point (15) on the solidsurface (7) whose surface is more strongly wetted by the analyte thanthe surrounding solid surface (7).
 8. The titration method according toclaim 7, wherein the reservoir drop (3) is applied to an anchor point(16) on the surface (7) of the solid (5), whose surface is more stronglywetted by the titrant than its surrounding solid surface.
 9. Thetitration method according to claim 7, wherein the titration quantity(17) of the titrant is guided from the reservoir drop (3) of the titrantapplied to the solid surface (7) on a path (18) to the analyte drop (1),where at least part of the surface of the path from the titrant isbetter wetted than the surface surrounding the path.
 10. The titrationmethod according to claim 9, wherein the titration quantity (17) of thetitrant is connected from the reservoir drop (3) to the analyte drop (1)over a path (18), which is connected to the anchor point (16) and/or theanalysis point (15), wherein the connection (14, 12) comprises a regionwhich is so narrow that, as a result of its surface tension, thereservoir drop (3) on the anchor point (16) does not leave the anchorpoint (16) without the action of an external force.
 11. The titrationmethod according to claim 4, wherein the reservoir drop (3) is moved atleast once over at least one region (41) of the surface (7), which ismore strongly wetted by the liquid than the surrounding surface, whereinthe area of this region (41) is smaller than the contact area of thereservoir drop (3) with the surface in order to separate a titrationquantity (17) from the reservoir drop (3).
 12. The titration methodaccording to claim 1, which is carried out in a climatic chamber tocontrol the thermodynamic boundary conditions.
 13. The titration methodaccording to claim 1, wherein a surface acoustic wave is launched in thedirection (10) of the analysis point (15) during and/or after thereaction between titrant and analyte (1) and the change in one or aplurality of parameters of the surface acoustic wave as a result of theinteraction with the liquid at the analysis point (15) is measured. 14.The titration method according to claim 1, wherein the reaction heat isdetermined during the reaction between titrant and analyte.
 15. Thetitration method according to claim 1, wherein the characteristicquantity in step c) comprises the electrical conductivity.
 16. Thetitration method according to claim 1, wherein the characteristicquantity in step c) comprises a colour change.
 17. The titration methodaccording to claim 1, wherein the characteristic quantity in step c)comprises the pH.
 18. The titration method according to claim 3, whereinone or a plurality of interdigital transducers (9, 11, 13) on apiezoelectric solid surface (7) are used to generate the surfaceacoustic wave or surface acoustic waves with a direction of emission ofthe surface acoustic waves in the direction of the desired momentumtransfer.
 19. The titration method according to claim 4, wherein one ora plurality of interdigital transducers (9, 11, 13) on a piezoelectricsolid surface (7) are used to generate the surface acoustic wave orsurface acoustic waves with a direction of emission of the surfaceacoustic waves in the direction of the desired momentum transfer. 20.The titration method according to claim 5, wherein one or a plurality ofinterdigital transducers (9, 11, 13) on a piezoelectric solid surface(7) are used to generate the surface acoustic wave or surface acousticwaves with a direction of emission of the surface acoustic waves in thedirection of the desired momentum transfer.